Bombyx mori BmSCP1 gene, its recombinant expression vector and application

Abstract

The present invention relates to the Bombyx mori BmSCP1 gene, its recombinant expression vector and application. Its nucleotide sequence is shown in SEQ ID NO.3. BmSCP1 is specifically expressed in the midgut of Bombyx mori and secreted into the intestinal fluid. This gene is up-regulated by the induction of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The increased expression of BmSCP1 can significantly inhibit the proliferation of BmCPV virus, indicating that this gene is a key gene affecting the virus resistance of Bombyx mori, and can provide a theoretical basis and target gene for the cultivation of transgenic resistant varieties of Bombyx mori.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to the Bombyx mori BmSCP1 gene and protein, and also relates to a recombinant expression vector containing the Bombyx mori BmSCP1 gene, as well as the applications of the Bombyx mori BmSCP1 gene and protein. Background Art

[0002] The silkworm Bombyx mori is an important economic insect, and silk is an important raw material for the silk industry. In many rural areas of our country, the sericulture industry is the pillar industry of the local economy and the main source of economic income for farmers. The silk industry generates hundreds of billions of yuan in income for sericulturists every year. However, the silkworm is facing a serious disease threat, and the losses caused by silkworm diseases account for about 20% of the total income of the sericulture production every year. Viral diseases are the most serious type of diseases threatening the sericulture industry, mainly caused by the Bombyx mori cytoplasmic polyhedrosis virus BmCPV, etc.

[0003] Due to the serious harm of BmCPV in sericulture production, scientific research workers have been hoping to find out the key genes affecting the virus resistance of silkworms, and then improve the virus resistance of silkworms through molecular biotechnology. Cloning and identifying the full-length sequences of the key genes affecting the virus resistance of silkworms has important theoretical value and practical significance for clarifying the mechanism of silkworms' resistance to viruses and cultivating resistant varieties for application in sericulture production. Summary of the Invention

[0004] In view of this, one of the purposes of the present invention is to provide the Bombyx mori BmSCP1 gene; the second purpose of the present invention is to provide a recombinant expression vector containing the Bombyx mori BmSCP1 gene; the third purpose of the present invention is to provide the application of the Bombyx mori BmSCP1 gene in the preparation of Bombyx mori varieties resistant to BmCPV virus; the fourth purpose of the present invention is to provide the application of the Bombyx mori BmSCP1 gene in the preparation of Bombyx mori varieties resistant to BmCPV virus; the fifth purpose of the present invention is to provide the application of the Bombyx mori BmSCP1 gene in the preparation of drugs for inhibiting the proliferation of BmCPV virus; the sixth purpose of the present invention is to provide the Bombyx mori BmSCP1 protein; the seventh purpose of the present invention is to provide the application of the Bombyx mori BmSCP1 protein in the preparation of drugs for reducing the infection mortality rate of BmCPV virus; the eighth purpose of the present invention is to provide the application of the Bombyx mori BmSCP1 protein in the preparation of drugs for inhibiting the proliferation of BmCPV virus.

[0005] To achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0006] 1. The Bombyx mori BmSCP1 gene, and the nucleotide sequence of the Bombyx mori BmSCP1 gene is as shown in SEQ ID NO.3.

[0007] 2. A recombinant expression vector containing the Bombyx mori BmSCP1 gene.

[0008] Preferably, the expression vector is obtained by inserting the sequence shown in SEQ ID NO.3 into the BamH I and Not I restriction sites of the psl1180 vector containing the midgut-specific promoter P3P + 5UI and the SV40 termination signal sequence.

[0009] 3. Use of the Bombyx mori BmSCP1 gene in the preparation of Bombyx mori varieties resistant to BmCPV virus.

[0010] 4. Use of the Bombyx mori BmSCP1 gene in the preparation of a drug for reducing the mortality rate of BmCPV virus infection.

[0011] 5. Use of the Bombyx mori BmSCP1 gene in the preparation of a drug for inhibiting the proliferation of BmCPV virus.

[0012] 6. Bombyx mori BmSCP1 protein, the amino acid sequence of the Bombyx mori BmSCP1 protein is shown in SEQ ID NO.4.

[0013] 7. Use of the Bombyx mori BmSCP1 protein in the preparation of a drug for reducing the mortality rate of BmCPV virus infection.

[0014] 8. Use of the Bombyx mori BmSCP1 protein in the preparation of a drug for inhibiting the proliferation of BmCPV virus.

[0015] In the present invention, h represents hours and min represents minutes.

[0016] The beneficial effects of the present invention are as follows: The present invention cloned the full-length CDS sequence of a key gene BmSCP1 that affects the resistance of Bombyx mori. This gene is specifically expressed in the midgut of Bombyx mori and secreted into the intestinal fluid, and is up-regulated by BmCPV virus. In order to study the function of this gene, using the full-length CDS sequence of this gene as a target, a transgenic overexpression vector was constructed. Through transgenic microinjection, transgenic positive individuals were screened. The transgenic Bombyx mori with overexpression of this gene showed significantly improved resistance to BmCPV virus. Therefore, this gene has important value in the research and application of transgenic disease-resistant breeding of Bombyx mori. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0018] Figure 1 Schematic diagram of the transgenic overexpression vector of the BmSCP1 gene.

[0019] Figure 2 qPCR detection of the expression level of BmSCP1 in transgenic Bombyx mori.

[0020] Figure 3Mortality statistics of transgenic silkworms infected with BmCPV.

[0021] Figure 4 Virus content detection of transgenic silkworms infected with BmCPV.

[0022] Figure 5 SDS-PAGE and western blot analysis of total intestinal fluid protein (A: SDS-PAGE analysis of total intestinal fluid protein; M: marker; 1: Intestinal fluid of SCP1-1 on the fourth day of the fifth instar; 2: Intestinal fluid of SCP1-2 on the fourth day of the fifth instar; 3: Intestinal fluid of control (WT) on the fourth day of the fifth instar; B: Western blot detection of total intestinal fluid protein; 1: Intestinal fluid of SCP1-1 on the fourth day of the fifth instar; 2: Intestinal fluid of SCP1-2 on the fourth day of the fifth instar; 3: Intestinal fluid of control (WT) on the fourth day of the fifth instar).

[0023] Figure 6 Virus amount in the midgut of silkworms after feeding BmCPV treated with intestinal fluid (A: Using the cDNA of the midgut of silkworm larvae at 24 h after infection as a template, qPCR was used to detect the expression level of the BmCPV genome fragment; B: After treating normal silkworm intestinal fluid with BmSCP1 antibody and PBS, incubating with the virus, feeding this virus to silkworms, and using the cDNA of the midgut of silkworm larvae at 24 h after infection as a template, qPCR was used to detect the expression level of the BmCPV genome fragment). Detailed implementation mode

[0024] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0025] Example 1: Cloning the full-length CDS sequence of the BmSCP1 gene

[0026] First, specific primers were designed according to the silkworm genome sequence. The forward primer of BmSCP1: 5'-atgattatcttacttgtattgg-3' (SEQ ID NO.1), and the reverse primer 5'-ttatgtatttgaaagccatcg-3' (SEQ ID NO.2). Using the midgut cDNA of silkworm strain Dazao (DZ) on the 3rd day of the 5th instar as a template for amplification, the PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes, then denaturation at 94°C for 40 seconds, annealing at 54°C for 40 seconds, extension at 72°C for 70 seconds, for a total of 30 cycles, and finally extension at 72°C for 10 minutes; the PCR product was identified by agarose gel electrophoresis and recovered, then ligated with the pMD19-T vector. The ligation reaction was carried out overnight at 16°C under the action of T4 DNA ligase, and then transformed into DH5α competent cells. After obtaining positive clones, they were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results showed that the CDS sequence of the BmSCP1 gene was successfully cloned.

[0027] Finally, the full-length CDS sequence of the BmSCP1 gene with 1440 bp was obtained. The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4, which contains 6 exons and 5 introns. Bioinformatics analysis showed that the first 16 amino acids of BmSCP1 were the signal peptide, and this protein had a peptidase_S28 domain.

[0028] Example 2: Construction of transgenic overexpression vector piggyBac[P3P+5UI-BmSCP1-SV40-3×p3DsRedafm]

[0029] To study the function of this gene, the expression level of this gene in silkworms was increased by overexpression, and then whether the virus proliferation changed was observed to prove the function of this gene.

[0030] Using the full-length CDS sequence of the BmSCP1 gene as the target, overexpression primers were designed. The upstream primer was BmSCP1oe-F: 5'- ggatcc atgattatcttacttgtattgg-3' (SEQ ID NO.5), and the downstream primer was BmSCP1oe-R: 5'– gcggccgc ttatgtatttgaaagccatcg-3' (SEQ ID NO.6). Using the cloned CDS plasmid of the BmSCP1 gene as the template for PCR amplification. The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes, then denaturation at 94°C for 40 seconds, annealing at 54°C for 40 seconds, extension at 72°C for 70 seconds, for a total of 29 cycles, and finally extension at 72°C for 10 minutes; the amplified target band was recovered, ligated, and transformed, and positive clones were screened and sequenced.

[0031] The plasmid successfully verified by sequencing was double-digested with BamH I and Not I, identified and recovered by agarose electrophoresis to obtain the BmSCP1 restriction fragment; at the same time, the psl1180 vector containing the midgut-specific promoter P3P+5UI and the SV40 termination signal sequence (see Liang Jiang. The 5'-UTR intron of the midgut-specific BmAPN4gene affects the level and location of expression in transgenic silkworms. Insect Biochemistry and Molecular Biology 63 (2015) 2 1-6, specifically, P3P+5UI and SV40 were respectively connected to the psl1180 vector multiple cloning site) was double-digested with BamH I and Not I, identified and recovered by agarose electrophoresis to obtain the psl1180-P3P+5UI-SV40 restriction fragment. The BmSCP1 restriction fragment and the psl1180-P3P+5UI-SV40 restriction fragment were connected, and then transformed. The positive clones were screened to obtain the psl1180-P3P+5UI-BmSCP1-SV40 vector. The piggyBac[3×p3DsRed afm] vector and psl1180-P3P+5UI-BmSCP1-SV40 were cut with Asc I, respectively, and the target bands were recovered and connected and transformed. The positive clones were screened to obtain the piggyBac[P3P+5UI-BmSCP1-SV40-3×p3DsRedafm] vector (pb-SCP1). The results are as follows Figure 1 shown.

[0032] Example 3. Transgenic microinjection and screening of positive individuals

[0033] (1) The silkworm eggs of the Dazao (DZ) variety of Bombyx mori were soaked in acid (hydrochloric acid with a specific gravity of 1.073, a temperature of 46°C, and a time of 5 minutes) to release diapause, and then placed in a dark environment at 15°C and 85% humidity for about 30 days until hatching. The larvae were collected and raised in a standard environment (temperature: 25°C, humidity: 80%). After the moths emerged, the male and female moths were mated for 4 hours. The eggs laid after the pairs were separated were non-diapause eggs and were used for the next step of microinjection.

[0034] Mix the pb-SCP1 recombinant vector and the helper plasmid A3H for later use. Arrange the laid silkworm eggs neatly on a clean glass slide. At 2 hours after egg laying, inject the mixed plasmid solution into the DZ silkworm eggs using an Eppendorf microinjector. The total number of injected silkworm eggs is 189. Seal the eggs with non-toxic glue and then incubate them in an environment at 25°C and a relative humidity of 80% for about 10 days until hatching. Collect and rear the 17 newly hatched G0 generation silkworm larvae with mulberry leaves until they turn into moths. Through self-crossing or backcrossing, the G0 generation silkworm moths obtained a total of 11 moth circles of G1 generation silkworm eggs, using an electric macro-fluorescence microscope to observe the G1 embryos and screen to obtain 2 positive moth circles, and then subculture and expand to obtain the transgenic silkworm systems SCP1-1 and SCP1-2.

[0035] Example 4. Detection of antiviral ability of transgenic silkworms

[0036] Extract the midgut RNA of the fifth instar third day larvae of the transgenic systems SCP1-1 and SCP1-2 and the non-transgenic control (WT), and perform qPCR detection using the specific primers of BmSCP1 and the internal reference gene TIF-4A. The results are as Figure 2 shown. The results show that the expression level of BmSCP1 in the transgenic silkworms is significantly higher than that of the control.

[0037] Take the newly molted fourth instar larvae of SCP1-1, SCP1-2 and WT, and orally feed them with BmCPV virus at the semi-lethal dose, one larva at a time. Count the mortality rate until before mounting. The results are as Figure 3 shown. The results show that when almost all of the non-challenged controls survived, the mortality rates of SCP1-1, SCP1-2 and WT were 22%, 27% and 45% respectively. The mortality rates of SCP1-1 and SCP1-2 were reduced by 23% and 18% respectively compared to WT.

[0038] At 72 h after feeding with BmCPV virus, extract the midgut RNA of the silkworms and reverse transcribe it into cDNA. Perform qPCR detection using the specific primers of the BmCPV virus gene fragments S1, S2, S4, S5, S8 and S10, and use the silkworm housekeeping gene TIF-4A as the internal reference. Set the detection result of each gene in WT as 100%, and convert the values of the transgenic lines based on this standard. Statistically analyze the virus content after infection with BmCPV. The results are as Figure 4As shown in the figure. The results showed that the contents of BmCPV virus gene fragments S1, S2, S4, S5, S8, and S10 in SCP1-1 only accounted for 22%, 23%, 18%, 23%, 19%, and 16% of the control respectively; the contents of BmCPV virus gene fragments S1, S2, S4, S5, S8, and S10 in SCP1-2 only accounted for 22%, 33%, 21%, 19%, 23%, and 29% of the control respectively. The results indicated that overexpression of BmSCP1 could significantly improve the resistance of transgenic silkworms to BmCPV.

[0039] Example 5. BmSCP1 protein exerts antiviral function in intestinal fluid

[0040] Extract the intestinal fluid of transgenic silkworms and the control on the fourth day of the fifth instar. After measuring the protein content of the intestinal fluid of each strain, equal amounts of samples were loaded for SDS-PAGE electrophoresis analysis. The results of Coomassie brilliant blue staining showed that the loading amounts of SCP1-1, SCP1-2, and the control were basically the same. Western blot detection was performed using the BmSCP1 antibody, and the results were as Figure 5 shown. The results indicated that the content of BmSCP1 protein in the intestinal fluid of transgenic silkworms was significantly higher than that of the control.

[0041] On the fourth day of the fifth instar, extract equal amounts of intestinal fluid from transgenic silkworms and the control, incubate them with an equal amount of BmCPV virus respectively, then feed them to the fourth instar normal silkworms. After 24 h, extract the midgut RNA of the silkworms, and perform qPCR detection using the specific primers of BmCPV gene fragments S1, S2, S4, S5, S8, S10 and the silkworm internal reference gene TIF-4A. The results were as Figure 6 shown in A. The results showed that the virus content in the silkworms fed with the virus treated with the intestinal fluid of transgenic silkworms was significantly lower than that of the control.

[0042] Extract the intestinal fluid of normal silkworms on the fourth day of the fifth instar, divide it into two equal parts, treat them with BmSCP1 antibody and PBS respectively, then incubate them with an equal amount of BmCPV virus, and then feed them to the fourth instar silkworms. Extract the midgut RNA of the silkworms 24 h after virus infection for qPCR detection. The results were as Figure 6 shown in B. The results showed that the virus content in the silkworms fed with the virus treated with the BmSCP1 antibody was significantly higher than that of the control.

[0043] The above results showed that the transgenic silkworms overexpressing BmSCP1 significantly inhibited the proliferation of BmCPV virus, and the mortality rate after virus infection was significantly reduced; more interestingly, after blocking the BmSCP1 protein in the intestinal fluid with an antibody, the amount of virus entering the silkworms increased significantly, indicating that the BmSCP1 protein exerted antiviral function in the initial stage of infection. These results indicated that the BmSCP1 gene was a key gene affecting the resistance of silkworms and could be used as a target gene for transgenic disease-resistant breeding of silkworms in future molecular breeding work.

[0044] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and details without departing from the scope defined by the claims of the present invention. Sequence Listing <110> Southwest University <120> Bombyx mori BmSCP1 Gene, Its Recombinant Expression Vector and Application <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <400> 1 atgattatct tacttgtatt gg 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <400> 2 ttatgtattt gaaagccatc g 21 <210> 3 <211> 1440 <212> DNA <213> Bombyx mori <400> 3 atgattatct tacttgtatt gggaagtttc ttctgcttga tatgtgcgga acctcggggt 60 gtggaacccc caatacgggt agacctccaa ccacctgagg aaactcaaac tcgttctgca 120 agaaatgttc agcagaactg ggtcactatg ccagtcgatc attttgatcc tcaaaatacg 180 gaaacttttc aaatgcgttt tatgtacaac gaggaattct ttggtggaaa cggatatcca 240 attttcatcc ttgtgggagg tgaatggact attgttgaag gatggttgcg cgcgggtaac 300 atgttcgaaa tggccaggga aaaccgtgga tatcagatat acactgaaca tcggtattac 360 gggcaaacgt tgccttatcg agattttaca acagaaaacc tgcagtactt gaacgtggat 420 caagctcttg ccgatcttgc ttactttata aatgaaatga aaaaacaacc tcgattcgca 480 gacagtaagg ttattttata cggtggctca tacgctgcca atatggtttt atggtttaag 540 caacgctatc cccatttggt cgaaggaacg gtggcctcaa gtggcccgat attggcaaaa 600 gtggacttta ccggatattt agaagtagtt cacgaagctt tccttcttga agggggtgag 660 cagtgtatag caactattaa acaaggaata gaagatacaa ttttagcaat gcaaactgaa 720 gagggtaaac gagaattgga ggcggcttat agattatgta atcctataga ttacaacgac 780 cgttttgatc ttggatactt ctcaggattg ataagttggt cgttctcaac gtcggtgcag 840 caagcccgtc caggaacttt gctaactatt tgtaatgact tcgccagtga aacctacgga 900 gtgacgccga tgcaaaaaat aggcggttat attgctacta cacgttctct cggtaatagc 960 tgctggcata tggaatatca aggaattctt aatgcttact caggaaatac caattcacgt 1020 gcgtggtact accagacgtg tacagaatat ggatattatc aaacagctcc gacttctggc 1080 actgtatttg atcctctcgt ttggctcagt gtagaatttt atgtggacat ttgcaaacgt 1140 gtcttcgatg aacggttcga tgaggcgttc gtttatgatg cgatagatcg tgtaaattta 1200 atattcggtg gcctggaacc caaagtaaac aacactatca acattcatgg ctatattgac 1260 ccgtggcgtg ctcttggtgt ctatgataga gatttaacag aaacctcgcc gacatacacg 1320 gtcacaagag catctcattg tttcgacatg caaggttggc tccggacgga tactataaga 1380 atgacaaacg cacagcaggc agcaagacga ttggtggctc gatggctttc aaatacataa 1440 <210> 4 <211> 479 <212> PRT <213> Bombyx mori <400> 4 Met Ile Ile Leu Leu Val Leu Gly Ser Phe Phe Cys Leu Ile Cys Ala 1 5 10 15 Glu Pro Arg Gly Val Glu Pro Pro Ile Arg Val Asp Leu Gln Pro Pro 20 25 30 Glu Glu Thr Gln Thr Arg Ser Ala Arg Asn Val Gln Gln Asn Trp Val 35 40 45 Thr Met Pro Val Asp His Phe Asp Pro Gln Asn Thr Glu Thr Phe Gln 50 55 60 Met Arg Phe Met Tyr Asn Glu Glu Phe Phe Gly Gly Asn Gly Tyr Pro 65 70 75 80 Ile Phe Ile Leu Val Gly Gly Glu Trp Thr Ile Val Glu Gly Trp Leu 85 90 95 Arg Ala Gly Asn Met Phe Glu Met Ala Arg Glu Asn Arg Gly Tyr Gln 100 105 110 Ile Tyr Thr Glu His Arg Tyr Tyr Gly Gln Thr Leu Pro Tyr Arg Asp 115 120 125 Phe Thr Thr Glu Asn Leu Gln Tyr Leu Asn Val Asp Gln Ala Leu Ala 130 135 140 Asp Leu Ala Tyr Phe Ile Asn Glu Met Lys Lys Gln Pro Arg Phe Ala 145 150 155 160 Asp Ser Lys Val Ile Leu Tyr Gly Gly Ser Tyr Ala Ala Asn Met Val 165 170 175 Leu Trp Phe Lys Gln Arg Tyr Pro His Leu Val Glu Gly Thr Val Ala 180 185 190 Ser Ser Gly Pro Ile Leu Ala Lys Val Asp Phe Thr Gly Tyr Leu Glu 195 200 205 Val Val His Glu Ala Phe Leu Leu Glu Gly Gly Glu Gln Cys Ile Ala 210 215 220 Thr Ile Lys Gln Gly Ile Glu Asp Thr Ile Leu Ala Met Gln Thr Glu 225 230 235 240 Glu Gly Lys Arg Glu Leu Glu Ala Ala Tyr Arg Leu Cys Asn Pro Ile 245 250 255 Asp Tyr Asn Asp Arg Phe Asp Leu Gly Tyr Phe Ser Gly Leu Ile Ser 260 265 270 Trp Ser Phe Ser Thr Ser Val Gln Gln Ala Arg Pro Gly Thr Leu Leu 275 280 285 Thr Ile Cys Asn Asp Phe Ala Ser Glu Thr Tyr Gly Val Thr Pro Met 290 295 300 Gln Lys Ile Gly Gly Tyr Ile Ala Thr Thr Arg Ser Leu Gly Asn Ser 305 310 315 320 Cys Trp His Met Glu Tyr Gln Gly Ile Leu Asn Ala Tyr Ser Gly Asn 325 330 335 Thr Asn Ser Arg Ala Trp Tyr Tyr Gln Thr Cys Thr Glu Tyr Gly Tyr 340 345 350 Tyr Gln Thr Ala Pro Thr Ser Gly Thr Val Phe Asp Pro Leu Val Trp 355 360 365 Leu Ser Val Glu Phe Tyr Val Asp Ile Cys Lys Arg Val Phe Asp Glu 370 375 380 Arg Phe Asp Glu Ala Phe Val Tyr Asp Ala Ile Asp Arg Val Asn Leu 385 390 395 400 Ile Phe Gly Gly Leu Glu Pro Lys Val Asn Asn Thr Ile Asn Ile His 405 410 415 Gly Tyr Ile Asp Pro Trp Arg Ala Leu Gly Val Tyr Asp Arg Asp Leu 420 425 430 Thr Glu Thr Ser Pro Thr Tyr Thr Val Thr Arg Ala Ser His Cys Phe 435 440 445 Asp Met Gln Gly Trp Leu Arg Thr Asp Thr Ile Arg Met Thr Asn Ala 450 455 460 Gln Gln Ala Ala Arg Arg Leu Val Ala Arg Trp Leu Ser Asn Thr 465 470 475 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <400> 5 ggatccatga ttatcttact tgtattgg 28 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <400> 6 gcggccgctt atgtatttga aagccatcg 29

Claims

1. Application of Bombyx mori BmSCP1 protein in preparing medicine for inhibiting proliferation of BmCPV virus, It is characterized in that: The silkworm BmSCP1 The amino acid sequence of the protein is shown in SEQ ID NO.4.

Images